Protective Immunity Of Targeting Staphylococcus Aureus Surface Protein A(SasA)

Staphylococcus aureus, an important opportunistic pathogen, causes avariety of infections, including skin and soft tissue infections, pneumonia, sepsis, endocarditis, septic arthritis and bone infection. Due to the emergence and prevalence of multi-antibiotics resistant S.aureus strains(methicillin-resistant Staphylococcus aureus, MRSA), the antibiotics-based therapeutic methods are becoming unreliable and there is an urgent need to develop vaccines and antibodies(m Abs) against S.aureus infections. Many virulence factors of S. aureus have been tested as candidate targets for immunotherapeutic strategies. However, not a single vaccine or antibody against S.aureus showed efficacy in human. Therefore, it is essential to focus on other antigens of S.aureus. Sas A, a surface protein of S.aureus, belongs to the serine-rich repeat proteins(SRRPs) family. Previous studies implied that Sas A mainly functions as an adhesion. Here, we showed that both Sas A vaccines and a monoclonal antibody(m Ab) targeting Sas A protect against S.aureus sepsis and peritonitis in Mice. Given the investigation of molecular epidemiology of Sas A and bioinformatics analysis, we confirmed that Sas A is a novel candidate target for immunotherapeutic measures against S.aureus infections.Bioinformatics and molecular epidemiology analysis showed that Sas A is prevalent among clinical S.aureus strains and there is a conserved region located in Sas A protein sequence. We screened 36 whole-genome sequences of S. aureus strains that are publicly available. These strains were isolated from a variety of sources and are representatives among S.aureus population. We found that all of these strains carry the Sas A gene. Moreover, we isolated 42 S.aureus strains from 306 Hospital of People’s Liberation Army.The results showed that 90%(24/27) of ST239(the most prevalent S.aureus strain in China) carry the Sas A gene. Sas A is composed of 2,271 amino acids. We analyzed the full-lengthamino acid sequence of Sas A with bioinformatics tools(Motif scan). Two serine-rich repeatregions(SRR) were found, and a non-repeat region(NRR) was found between these SRRs. The amino acid sequences of SRR1, NRR and SRR2 represent 48-229 aa, 230-751 aa and 752-2213 aa of full-length Sas A, respectively. Next, we conducted the sequence homology analysis. SRR1, NRR and SRR2 display 75.8%, 90.4% and 49.3% identity at the amino acid level, respectively, suggesting that NRR is the most conserved region of Sas A. In addition, we obtained serum samples isolated from patients infected with S.aureus and healthy volunteers. ELISA results showed that Ig G levels against Sas A in serum obtained from patients infected with S. aureus were higher than those obtained from healthy individuals, indicating that Sas A is expressed during S. aureus infections in vivo and Sas A is able to induce antibody response in humans.Active immunization with Sas A is able to confer protection. We prepared different recombinant fragments of Sas A, namely SRR1(48-229 aa), NRR(230-751 aa), NRR1(230-540 aa) and NRR2(490-751aa). SRR1, NRR1 and NRR were highly immunogenic in mice and induced high-level antibody response. Immunization with SRR1 and NRR1 generated protective immunity against lethal peritoneal challenge with S. aureus USA300. NRR and NRR2 immunization were not able to confer protective immunity. Moreover, passive immunization with murine polyclonal antibody against SRR1-NRR(48-540 aa) can also provide protection against lethal peritoneal challenge with S. aureus, suggesting that humoral immunity plays an important role in Sas A protective immunity.Passive immunization with an anti-Sas A monoclonal antibody is able to confer protection. Besides active immunization with vaccines, passive immunization with antibodies is another important method to combat S.aureus infections. MAbs are attractive antibody-based therapeutic reagents because of their homogeneity, stability and low-immunogenicity. By using hybridoma technology and ELISA screen, we prepared high-titer m Abs targeting different epitopes of Sas A. We evaluated their efficacy in vitro and in vivo and selected 2H7 for further studies.2H7 binds with the conserved domain of Sas A, recognizes S.aureus and promotes opsonophagocytic killing of S. aureus by neutrophils. The light chain and heavy chain genes of 2H7 were amplified by 5’ RACE, reverse transcription and nested PCR. Sequences were analyzed and the variable region, frame region and complementary determining region of 2H7 were obtained. The ELISA data showed that 2H7 did not recognize SRR1 but bound with high affinity to NRR1 and NRR, indicating that 2H7 recognizes the conserved domain of Sas A. Furthermore, 2H7 also displayed the binding activity to native Sas A expressed on the surface of wild-type S. aureus. A previous study showed that Sas A(90-723 aa) can bind to human platelets and contribute to endocarditis in an animal model. We confirmed that NRR but not SRR1 was able to bind mouse platelets by using flow cytometry. However, the addition of 2H7 did not interfere with the association between NRR and mouse platelets, suggesting that 2H7 may not provide protection in a staphylococcal endocarditis model. Neutrophils play an important role in the front-line of host-pathogen battle and antibodies function as opsonins and promote the phagocytosis of pathogen byneutrophils. In both an ex vivo whole-blood killing model and human neutrophils HL-60 model, 2H7 significantly promote opsonophagocytic killing of S. aureus.2H7 conferred protection in murine S.aureus infectons models. As chronic dialysis patients are vulnerable to MRSA infections, we evaluated the efficacy of 2H7 in both a murine sepsis model and a murine peritoneal infection model. We chose USA300 and ST239 as the challenging strains, which are prevalent isolates in North America and Asian regions, respectively. In a murine sepsis model, 24 h before lethal challenge with USA300 and ST239, passive immunization with 2H7 significantly improved mice survival. In addition, 2H7 was administrated 1 h post challenge with USA300 and showed efficacy, suggesting that 2H7 has the therapeutic potential. In a peritoneal infection model, 2H7 prophylaxis also conferred protection against lethal challenges with USA300 and ST239. As chronic dialysis patients are not likely to be exposed to a large amount of S.aureus, we also evaluated the efficacy of 2H7 in sublethal challenge models. 2H7 prophylaxis was able to offer protection against staphylococcal abscess lesions in the peritoneal infection model. Of note, in both the sepsis model and the peritoneal infection model, passive immunization of 2H7 can reduce the staphylococcal loads in kidneys.In conclusion, Sas A is prevalent among S.aureus strains and composed of a conserverd region. Active immunization with Sas A and passive immunization with an anti-Sas A monoclonal antibody are able to confer protection against MRSA infections in mice. All in all, Sas A is a novel candidate target to combat agsinst MRSA infections.